Method for attachment of silylated molecules to glass surfaces

ABSTRACT

A method for the efficient immobilization of silylated molecules such as silylated oligonucleotides or proteins onto unmodified surfaces such as a glass surface is provided. Also provided are compounds, devices, and kits for modifying surfaces such as glass surfaces.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional application No.60/383,564, filed May 28, 2003, which is incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

Surface modification plays an important role in micro-array biomoleculedetection technology for controlling backgrounds and spot morphology.Several modifications were developed using different type ofcommercially available silanes such as silyl amines, aldehydes, thiolsetc. for immobilization of biomolecules such as oligonucleotides. Aftercoating the surface with reactive silanes, the next challenge isimmobilization of required biomolecules on the modified surface. Thesurface loadings always vary with different silanes and even same silanemay not give reproducible results. Reproducibility of optimum surfaceloading has always been a great challenge in this field since surfaceloading dictates the performance of the assay. Even with simple linearmolecules for immobilization, the optimum loading on the surface isdifficult to achieve.

Attaching DNA to a modified glass surface is a central step for manyapplications in DNA diagnostics industry including gene expressionanalysis. In general, DNA can be attached to a glass surface eitherthrough non-covalent, ionic interactions, or through multi-stepprocesses or simple coupling reactions. Several methods have beenreported in the literature using glass surface modified with differenttypes of silylating agents¹⁻⁶. All these reported methods involvesilylating step which uses expensive reagents and analytical tools.Also, these methods are also multi-step processes that are laborintensive and expensive⁸⁻⁹. Earlier reported methods have involved alaborious synthesis and time consuming procedure⁷. Indeed, many of thecurrent immobilization methods suffer from one or more of a number ofdisadvantages. Some of these are, complex and expensive reaction schemeswith low oligonucleotide loading yields, reactive unstable intermediatesprone to side reactions and unfavorable hybridization kinetics of theimmobilized oligonucleotide. The efficient immobilization ofoligonucleotides or other molecules on glass surface in arrays requiresa) simple reliable reactions giving reproducible loading for differentbatches, b) stable reaction intermediates, c) arrays with high loadingand fast hybridization rates, d) high temperature stability, e) lowcost, f) specific attachment at either the 5′- or 3′-end or at aninternal nucleotide and g) low background.

The present invention represents a significant step in the direction ofmeeting or approaching several of these objectives.

SUMMARY OF THE INVENTION

The present invention fulfills the need in the art for methods for theattachment of molecules such as oligonucleotides onto unmodifiedsurfaces such as a glass surface without the need for laborioussynthetic steps, with increase surface loading densities, and withgreater reproducibility and which avoids the need for pre-surfacemodifications. Molecules such as DNA can be silylated at either the 3′or 5′ ends as discussed below and the 3′ or 5′-silylated DNA may then becovalently attached directly to a surface such as a pre-cleaned glasssurface (Scheme) for use in hybridization assays. Furthermore, thoroughthe use of certain silylating reagents, it is now possible to furtherenhance surface loading densities by using modified silylating agentshaving multiple molecules attached thereto. The present invention thusprovides novel methods for attaching molecules onto a substrate, devicesprepared by such methods, and compositions. This method provides greatadvantages over the present technology in terms of simplicity, cost,speed, safety, and reproducibility.

Thus, in one embodiment of the invention, a method is provided forimmobilizing a molecule onto a surface, said method comprising the stepsof:

(a) contacting the molecule with an agent so as to form a reactiveintermediate, said agent having a formula i:(R₁)(R₂)(R₃)Si—X—NCY  iwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; and Y represents oxygen or sulfur, with theproviso that at least one of R₁, R₂ or R₃ represents C₁-C₆ alkoxy; and

(b) contacting the reactive intermediate with said surface so as toimmobilize the molecule onto said surface.

In one aspect of this embodiment, a method is provided for immobilizinga molecule onto a glass surface.

In another embodiment of the invention, a method is provided forimmobilizing a molecule onto a surface, said method comprising the stepsof:

(a) contacting Si(NCY)₄ wherein Y represents oxygen or sulfur with anagent so as to form a first reactive intermediate, said agent having aformula ii:(R₁)(R₂)(R₃)Si—X-Z  iiwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; and Z represents a hydroxy or amino group,with the proviso that at least one of R₁, R₂ or R₃ represents C₁-C₆alkoxy;

(b) contacting the first reactive intermediate with a molecule so as toform a second reactive intermediate;

(c) contacting the second reactive intermediate with said surface so asto immobilized the molecule onto said surface. The method allows for theproduction of branched captured molecules structures such as branchedoligonucleotides on a surface which is useful for enhancing detection oftarget analytes such as nucleic acids.

In one aspect of this embodiment of the invention, a method is providedfor immobilizing a molecule onto a glass surface.

In another embodiment of the invention, a compound is provided havingthe formula iii:(R₁)(R₂)(R₃)Si—X—NHCYL-M  iiiwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; Y represents oxygen or sulfur; L representsa linking group; and M represents a molecule, with the proviso that atleast one of R₁, R₂, or R₃ represent C₁-C₆ alkoxy.

In another embodiment of the invention, a compound is provided having aformula iv:(R₁)(R₂)(R₃)Si—X-Z-CYNH—Si(NCY)₃  ivwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; Y represents oxygen or sulfur; and Zrepresents oxygen or NH, with the proviso that at least one of R₁, R₂,or R₃ represents C₁-C₆ alkoxy.

In another embodiment of the invention, a compound is provided having aformula v:(R₁)(R₂)(R₃)Si—X-Z-CYNH—Si(NHCYL-M)₃  vwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; Y represents oxygen or sulfur; L representsa linking group; and Z represents oxygen or NH; and M represents amolecule, with the proviso that at least one of R₁, R₂, or R₃ representC₁-C₆ alkoxy.

In another embodiment of the invention, a compound is provided having aformula vi:((R₁)(R₂)(R₃)Si—X-Z-CYNH)₂-Si(NCY)₂  viwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; Y represents oxygen or sulfur; and Zrepresents oxygen or NH, with the proviso that at least one of R₁, R₂,or R₃ represents C₁-C₆ alkoxy.

In another embodiment of the invention, a compound is provided having aformula vii:((R₁)(R₂)(R₃)Si—X-Z-CYNH)₂Si(NHCYL-M)₂  viiwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; L represents alinking group; X represents linear or branched C₁-C₂₀ alkyl or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or moreheteroatoms comprising oxygen, nitrogen, or sulfur; Y represents oxygenor sulfur; and Z represents oxygen or NH; and M represents a molecule,with the proviso that at least one of R₁, R₂, or R₃ represents C₁-C₆alkoxy.

In another embodiment of the invention, kits are provided for preparingmodified substrates. The kits may include reagents for silyatingmolecules and optional substrates.

These and other embodiments of the invention will become apparent inlight of the detailed description below.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme that illustrates one embodiment of the invention. Thescheme shows the modification of a molecule such as an oligonucleotidemodified at either a 3′-amino or 5′-amino to produce a silylated DNAintermediate. This silylated intermediate is then spotted onto a surfaceof a substrate, e.g., glass substrate and washed.

FIG. 2 illustrates spot morphology after spotting a substrate with a DMFsolution containing a silylated DNA in water or DMF. Branching andspreading of the spot was observed with the aqueous solution.

FIG. 3 illustrates spot morphology using a DMF solution containing asilyated DNA spotted on a overhydrated substrate. Branching of the spotwas observed with the over hydrated substrate.

FIG. 4 illustrates spot morphology with an aqueous solution containingno silylated DNA (blank control) and with silylated DNA (silyl).

FIG. 5 is (a) a scheme that illustrates another embodiment of theinvention. The scheme shows the coupling of a tetraisocyanatosilane witha 1-amino-4-triethoxysilylbenzene to form a first reactive intermediate4. The reactive intermediate is then coupled to a oligonucleotide havinga free 3′ or 5′-amino group to silylated DNA intermediate as a secondreactive intermediate containing three molecules bound thereto. Thissilylated intermediate is then spotted onto a surface of a substrate,e.g., glass substrate. In part (b), a scheme is provided thatillustrates another embodiment of the invention. The scheme shows thecoupling of a tetraisocyanatosilane with a1-amino-4-triethoxysilylbenzene to form a first reactive intermediate 4.The reactive intermediate is then coupled to a oligonucleotide having afree 3′ or 5′-amino group to silylated DNA intermediate as a secondreactive intermediate containing two molecules bound thereto.

FIG. 6 illustrates the results of detection of M13 capture sequencesusing a DNA array chip prepared as described in Example 1 (method no.1). In plate no. 1, a non-complementary nanoparticle-labeledoligonucleotide probe was used. In plates nos. 2 and 3, a specificcomplementary nanoparticle-labeled oligonucleotide probe was used. Asexpected, the plates using the specific complementary probes showeddetection events. See Example 3.

FIG. 7 illustrates the results of detection of Factor V target sequenceusing a sandwich hybridization assay. A DNA array chip was prepared asdescribed in Example 1 (method no. 1) using Factor V capture probe. TheDNA chip performed as expected. See Example 4.

FIG. 8 illustrates the results of detection of MTHFR target sequenceusing a DNA array chip prepared as described in Example 1 (method no.1). The DNA chip performed as expected. Plate No. 1 shows that thedetection probe does not hybridized above its melting temperature. PlateNo. 2 showed detection of a 100 mer MTHFR synthetic target. Plate No. 3showed detection of a MTHFR PCR product. See Example 5. See Example 5.

FIG. 9 illustrates the results of detection of Factor V target sequenceusing a DNA array chip prepared as described in Example 1 (method no.1). The DNA chip performed as expected. No non-specific background noisewas observed. See Example 6.

FIG. 10 illustrates the results of detection of Factor V target sequenceusing a DNA array chip prepared as described in Example 1 (method no.1). The DNA chip performed as expected. The probes reacted specificallyto the target sequence and no cross-hybridization between the probes andtargets was observed. See Example 7.

DESCRIPTION OF THE INVENTION

All patents, patent applications, and references cited herein areincorporated by reference in their entirety.

As defined herein, the term “molecule” refers to any desired specificbinding member that may be immobilized onto the surface of thesubstrate. The “specific binding member,” as defined herein, meanseither member of a cognate binding pair. A “cognate binding pair,” asdefined herein, is any ligand-receptor combination that willspecifically bind to one another, generally through non-covalentinteractions such as ionic attractions, hydrogen bonding, Vanderwaalsforces, hydrophobic interactions and the like. Exemplary cognate pairsand interactions are well known in the art and include, by way ofexample and not limitation: immunological interactions between anantibody or Fab fragment and its antigen, hapten or epitope; biochemicalinteractions between a protein (e.g. hormone or enzyme) and its receptor(for example, avidin or streptavidin and biotin), or between acarbohydrate and a lectin; chemical interactions, such as between ametal and a chelating agent; and nucleic acid base pairing betweencomplementary nucleic acid strands; a peptide nucleic acid analog whichforms a cognate binding pair with nucleic acids or other PNAs. Thus, amolecule may be a specific binding member selected from the groupconsisting of antigen and antibody-specific binding pairs, biotin andavidin binding pairs, carbohydrate and lectin bind pairs, complementarynucleotide sequences, complementary peptide sequences, effector andreceptor molecules, enzyme cofactor and enzymes, and enzyme inhibitorsand enzymes. Other specific binding members include, without limitation,DNA, RNA, polypeptide, antibody, antigen, carbohydrate, protein,peptide, amino acid, carbohydrate, hormone, steroid, vitamin, drug,virus, polysaccharides, lipids, lipopolysaccharides, glycoproteins,lipoproteins, nucleoproteins, oligonucleotides, antibodies,immunoglobulins, albumin, hemoglobin, coagulation factors, peptide andprotein hormones, non-peptide hormones, interleukins, interferons,cytokines, peptides comprising a tumor-specific epitope, cells,cell-surface molecules, microorganisms, fragments, portions, componentsor products of microorganisms, small organic molecules, nucleic acidsand oligonucleotides, metabolites of or antibodies to any of the abovesubstances. Nucleic acids and oligonucleotides comprise genes, viral RNAand DNA, bacterial DNA, fungal DNA, mammalian DNA, cDNA, mRNA, RNA andDNA fragments, oligonucleotides, synthetic oligonucleotides, modifiedoligonucleotides, single-stranded and double-stranded nucleic acids,natural and synthetic nucleic acids. Preparation of antibody andoligonucleotide specific binding members is well known in the art. Themolecules (M) have at least one or more nucleophilic groups, e.g.,amino, carboxylate, or hydroxyl, that are capable of linking or reactingwith the silylating agents to form a reactive silylated molecule whichis useful for modifying the surfaces of substrates. These nucleophilicgroups are either already on the molecules or are introduced by knownchemical procedures.

As defined herein, the term “substrate” refers any solid supportsuitable for immobilizing oligonucleotides and other molecules are knownin the art. These include nylon, nitrocelluose, activated agarose,diazotized cellulose, latex particles, plastic, polystyrene, glass andpolymer coated surfaces. These solid supports are used in many formatssuch as membranes, microtiter plates, beads, probes, dipsticks, opticalfibers, etc. Of particular interest as background to the presentinvention is the use of glass and nylon surfaces in the preparation ofDNA microarrays which have been described in recent years (Ramsay, Nat.Biotechnol., 16: 40-4 (1998)). The journal Nature Genetics has publisheda special supplement describing the utility and limitations ofmicroarrays (Nat. Genet., 21(1): 1-60 (1999). Typically the use of anysolid support requires the presence of a nucleophilic group to reactwith the silylated molecules of the invention that contain a “reactivegroup” capable of reacting with the nucleophilic group. Suitablenucleophilic groups or moieties include hydroxyl, sulfhydryl, and aminogroups or any moiety that is capable of coupling with the silyatedmolecules of the invention. Chemical procedures to introduce thenucleophilic or the reactive groups onto solid support are known in theart, they include procedures to activate nylon (U.S. Pat. No.5,514,785), glass (Rodgers et al., Anal. Biochem., 23-30 (1999)),agarose (Highsmith et al., J., Biotechniques 12: 418-23 (1992) andpolystyrene (Gosh et al., Nuc. Acid Res., 15: 5353-5372 (1987)). Thepreferred substrate is glass.

The term “analyte,” or “target analyte”, as used herein, is thesubstance to be quantitated or detected in the test sample using devicesprepared by the method of the present invention. The analyte can be anysubstance for which there exists a naturally occurring specific bindingmember (e.g., an antibody, polypeptide, DNA, RNA, cell, virus, etc.) orfor which a specific binding member can be prepared, and the analyte canbind to one or more specific binding members in an assay.

In one embodiment of the invention, a method is provided forimmobilizing a molecule onto a substrate surface, said method comprisingthe steps of contacting the molecule with an agent so as to form areactive intermediate, said agent having a formula i:(R₁)(R₂)(R₃)Si—X—NCY  iwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; and Y represents oxygen or sulfur, with theproviso that at least one of R₁, R₂ or R₃ represents C₁-C₆ alkoxy; andcontacting the reactive intermediate with said surface so as toimmobilized the molecule onto said surface.

In practice, the molecule is contacted with the agent in solution.Generally, the molecule is dissolved in a solution and agent is addeddrop-wise to the molecule solution. Suitable, but non-limiting, examplesof solvents used in preparing the solution include DMF, DMSO, ethanoland solvent mixtures such as DMSO/ethanol. The preferred solvent isethanol. Water is preferably excluded from the reaction solvent becausewater may interfere with the efficient modification of the molecule.However, if water is necessary to increase solubility of the molecule inthe solution, the amount of water generally ranges from about 0.1% toabout 1%, usually no greater than 1%.

The amount of molecule to agent generally ranges from about 1 to about1.5 typically from about 1 to about 1.1, preferably from about 1 toabout 1 molar equivalents. The reaction may be performed in any suitabletemperature. Generally, the temperature ranges between about 0° C. andabout 40° C., preferably from about 20° C. to about 25° C. The reactionis stirred for a period of time until sufficient amount of molecule andagent reacts to form a reactive intermediate. The reactive intermediatehas a structure defined by formula iii.

Thereafter, the reaction solution containing the reactive intermediateis then concentrated and dissolved in desired solvent to provide aspotting solution which is then applied to the surface of a substrate.The reactive intermediate is applied as a spotting solution. Anysuitable solvent may be used to prepare the spotting solution. Suitable,but non-limiting, examples of solvents used in preparing the spottingsolution include DMF, DMSO, and ethanol as well as any suitable solventmixtures such as DMF/pyridine. Any suitable concentration of thespotting solution may be prepared, generally the concentration of thespotting solution is about 1 mM. Any suitable spotting technique may beused to produce spots. Representative techniques include, withoutlimitation, manual spotting, ink-jet technology such as the onesdescribed in U.S. Pat. Nos. 5,233,369 and 5,486,855; array pins orcapillary tubes such as the ones described in U.S. Pat. Nos. 5,567,294and 5,527,673; microspotting robots (e.g., available from Cartesian);chipmaker micro-spotting device (e.g., as available from TeleChemInterational). Suitable spotting equipment and protocols arecommercially available such as the ArrayIt® chipmaker 3 spotting device.The spotting technique can be used to produce single spots or aplurality of spots in any suitable discrete pattern or array.

In the preferred embodiment, the agent is triethoxysilylisocyanate. Thepreferred molecule is a nucleic acid.

In another embodiment of the invention, a method is provided forimmobilizing a molecule onto a substrate surface, said method comprisingthe steps of contacting Si(NCY)₄ with an agent so as to form a firstreactive intermediate, said agent having a formula ii:(R₁)(R₂)(R₃)Si—X-Z  iiwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; wherein Y represents oxygen or sulfur; andZ represents a hydroxy or amino group, with the proviso that at leastone of R₁, R₂ or R₃ represents C₁-C₆ alkoxy; contacting the firstreactive intermediate with a molecule so as to form a second reactiveintermediate; and contacting the second reactive intermediate with saidsurface so as to immobilized the molecule onto said surface.

In this embodiment of the invention, the method provide for amodification of substrate surfaces with branched molecules so as toincrease molecule loading on the substrate surface. These branchedmolecules behave like dendrimers to enhance sensitivity in assayperformance. In practice, either Si(NCO)₄ or Si(NCS)₄ are reacted with acompound of formula ii to form a first reactive intermediate having theformula iv:(R₁)(R₂)(R₃)Si—X-Z-CYNH—Si(NCY)₃  ivwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; Y represents oxygen or sulfur; and Zrepresents oxygen or NH, with the proviso that at least one of R₁, R₂,or R₃ represents C₁-C₆ alkoxy.

Generally, Si(NCO)₄ or Si(NCS)₄ is dissolved in a suitable dry solventas described above. In practice, ethanol is the preferred solvent. Theresulting ethanol solution is contained in a reaction flask and asolution of formula ii compound is added to the reaction flask. Theformula ii solution may include any of the dried solvents describedabove. In practice, ethanol is the preferred solvent. The reactiontemperature generally ranges from about 0° C. to about 40° C.,preferably about 22° C. The reaction mixture is allowed to stir fromabout 1 min to about 60 min, usually about 5 min to about 10 min, untilit reaches completion. The molar amount of Si(NCO)₄ or Si(NCS)₄ toformula ii compound generally ranges from about 3:1 to 1:1, preferablyabout 1:1.

Thereafter, the molecule is contacted with the first reactiveintermediate to form a second reactive intermediate having the formulav:(R₁)(R₂)(R₃)Si—X-Z-CYNH—Si(NHCYL-M)₃  vwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; L represents a linking group; Y representsoxygen or sulfur; and Z represents oxygen or NH; and M represents amolecule, with the proviso that at least one of R₁, R₂, or R₃ representC₁-C₆ alkoxy. The linking group L may be a nucleophile that is naturallypresent or chemically added to the molecule such as an amino, sulfhydrylgroup, hydroxy group, carboxylate group, or any suitable moiety. L mayrepresent —NH, —S—, —O—, or —OOC—.

The molecule is contacted with the first reactive intermediate insolution. Generally, the molecule is dissolved in a solvent and addeddropwise to the reaction flask containing the first reactiveintermediate. The molecule is generally mixed in any suitable solvent asdescribed above. The molar amount of molecule to first reactiveintermediate generally ranges from about 1 to about 10 typically fromabout 1 to about 3, preferably from about 1 to about 4. The reaction maybe performed in any suitable temperature. Generally, the temperatureranges between about 0° C. and about 40° C., preferably from about 20°C. to about 25° C. The reaction is stirred for a period of time untilsufficient amount of molecule and first reactive intermediate reacts toform a second reactive intermediate. Generally, an excess amount ofmolecule is used to react with the first reactive intermediate. Inpractice, typically at least 3 equivalents of molecule to 1 equivalentof first reactive intermediate is used.

Thereafter, the second reactive intermediate is then applied to thesurface of a substrate using techniques described above.

In another aspect of this invention, if the ratio of Si(NCO)₄ orSi(NCS)₄ to formula ii compound is about 1:2 equiv./equiv., a firstreactive intermediate is formed having the formula vi:((R₁)(R₂)(R₃)Si—X-Z-CYNH)₂-Si(NCY)₂  vi

wherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; Y represents oxygen or sulfur; and Zrepresents oxygen or NH, with the proviso that at least one of R₁, R₂,or R₃ represents C₁-C₆ alkoxy. Preferably, R₁, R₂ and R₃ representmethoxy, X represents phenyl, Y represents oxygen, and Z represents NH.

Thereafter, the molecule is contacted with the first reactiveintermediate of formula vi as described above to produce a secondreactive intermediate having the formula vii:((R₁)(R₂)(R₃)Si—X-Z-CYNH)₂Si(NHCYL-M)₂  vii

wherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; L represents alinking group; X represents linear or branched C₁-C₂₀ alkyl or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or moreheteroatoms comprising oxygen, nitrogen, or sulfur; Y represents oxygenor sulfur; and Z represents oxygen or NH; and M represents a molecule,with the proviso that at least one of R₁, R₂, or R₃ represent C₁-C₆alkoxy. The linking group L may be a nucleophile that is naturallypresent or chemically added to the molecule such as an amino, sulfhydrylgroup, hydroxy group, carboxylate group, or any suitable moiety. L mayrepresent —NH, —S—, —O—, or —OOC—. Generally, an excess amount ofmolecule is used to react with the first reactive intermediate. Inpractice, typically at least 3 equivalents of molecule to 1 equivalentof first reactive intermediate is used.

Thereafter, the second reactive intermediate is then applied to thesurface of a substrate using the techniques described above.

In another embodiment of the invention, a compound is provided havingthe formula iii:(R₁)(R₂)(R₃)Si—X—NHCYL-M  iiiwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; L represents alinking group; X represents linear or branched C₁-C₂₀ alkyl or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or moreheteroatoms comprising oxygen, nitrogen, or sulfur; Y represents oxygenor sulfur; and M represents a molecule, with the proviso that at leastone of R₁, R₂, or R₃ represent C₁-C₆ alkoxy. The linking group L may bea nucleophile that is naturally present or chemically added to themolecule such as an amino, sulfhydryl group, hydroxy group, carboxylategroup, or any suitable moiety. L may represent —NH, —S—, —O—, or —OOC—.In the preferred embodiment, R₁, R₂, and R₃ represent alkoxy, Lrepresents —NH—, X represents propyl, and Y represents O. The compoundis useful for modifying substrate surfaces with a desired molecule.

In another embodiment of the invention, a compound is provided having aformula iv:(R₁)(R₂)(R₃)Si—X-Z-CYNH—Si(NCY)₃  ivwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X representslinear or branched C₁-C₂₀ alkyl or aryl substituted with one or moregroups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆alkoxy, optionally substituted with one or more heteroatoms comprisingoxygen, nitrogen, or sulfur; Y represents oxygen or sulfur; and Zrepresents oxygen or NH, with the proviso that at least one of R₁, R₂,or R₃ represents C₁-C₆ alkoxy. In the preferred embodiment, R₁, R₂, andR₃ represent ethoxy or methoxy, X represents benzyl, Y representsoxygen, and Z represents NH. The compound is useful for modifyingmolecules so that they can be attached to substrate surfaces.

In another embodiment of the invention, a compound is provided having aformula v:(R₁)(R₂)(R₃)Si—X-Z-CYNH—Si(NHCYL-M)₃  vwherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆alkyl, phenyl, or aryl substituted with one or more groups selected fromthe group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; L represents alinking group; X represents linear or branched C₁-C₂₀ alkyl or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or moreheteroatoms comprising oxygen, nitrogen, or sulfur; Y represents oxygenor sulfur; and Z represents oxygen or NH; and M represents a molecule,with the proviso that at least one of R₁, R₂, or R₃ represent C₁-C₆alkoxy. The linking group L may be a nucleophile that is naturallypresent or chemically added to the molecule such as an amino, sulfhydrylgroup, hydroxy group, carboxylate group, or any suitable moiety. L mayrepresent —NH, —S—, —O—, or —OOC—. In the preferred embodiment, R₁, R₂,and R₃ represent methoxy or ethoxy, X represents 3- or 4-phenyl, Yrepresents oxygen, and Z represents NH. The compound is useful formodifying molecules so that they can be attached to substrate surfaces.

In another embodiment of the invention, a device is provided for thedetection of target analytes in a sample. The device comprises a surfacehaving an immobilized molecule as a specific binding member to thetarget analyte, wherein said surface is prepared by any of the abovemethods. The preferred surface is a glass surface. The surface may haveone or more different specific binding members attached thereto in anarray to allow for the detection of different portions of a targetanalyte or multiple different types of target analytes.

In another embodiment of the invention, a kit is provided. The kit maycomprise one or more containers containing any of the silylating agentsmentioned above with an optional substrate, and a set of instructions.

EXAMPLES

The invention is demonstrated further by the following illustrativeexamples. The examples are offered by way of illustration and are notintended to limit the invention in any manner. In these examples allpercentages are by weight if for solids and by volume if for liquids,and all temperatures are in degrees Celsius unless otherwise noted.

Example 1 Preparation of DNA Array Chips

This Example provides a general procedure for the covalent attachment ofa molecule, e.g., 3′ or 5′ silylated DNA, directly to surfaces such aspre-cleaned glass surface via single silylated molecule or dendriticsilylated molecule procedure.

(a) Method No. 1

As shown in FIG. 1, a method is shown for attaching a 3′-amino or5′-amino DNA molecule to a pre-cleaned glass surface. 3′-Amine linkedDNA is synthesized by following standard protocol for DNA synthesis onDNA synthesizer. The 3′ amine modified DNA synthesized on the solidsupport was attached through succinyl linker to the solid support. Aftersynthesis, DNA attached to the solid support was released by usingaqueous ammonia, resulting in the generation of a DNA strand containinga free amine at the 3′-end. The crude material was purified on HPLC,using triethyl ammonium acetate (TEAA) buffer and acetonitrile. Thedimethoxytrityl (DMT) group was removed on the column itself usingtriflouroacetic acid.

After purification, 1 equivalents of 3 ′-amine linked DNA wassubsequently treated with 1.2 equivalents of triethoxysilyl isocyanate(GELEST, Morrisville, Pa. USA) for 1-3 h in 10% DMSO in ethanol at roomtemperature. Traces of water that remained in the DNA followingevaporation did not effect the reaction. After 3 h, the reaction mixturewas evaporated to dryness and spotted directly on pre-cleaned glasssurface using an arrayer (Affymetrix, GMS 417 arrayer with 500 micronpins for spotting). Typically, 1 mM silylated DNA was used to array aglass surface and the arrayed substrate is then kept in the chamber for4 h-5 h. Thereafter, the slides were incubated in nanopure water for 10minutes to remove the unbound DNA, washed with ethanol, and dried in thedessicator. After drying, these plates were tested with target DNAsamples.

In a preliminary study using linear silyl oligonucleotides prepared bythe above procedure to spot a glass surface, it was observed thatspotting in DMSO or DMF medisurprisingly controlled spot branching ordiffusion. See FIG. 2. The spot morphology was clean and discrete. Ifthe substrate was overhydrated in the dessicator chamber prepared byfiling a portion of a chamber with water and storing the glass slides ona rack above the water level overnight, the slides become overhydrated.Undesirable branching of the spot was observed on overhydrated slides,even when DMSO or DMF solvent is used. See FIG. 3. When water was usedas the sole solvent for spotting, the resultant spots were branched outand spread to other spots. See FIG. 4. Without being bound to any theoryof operation, an aqueous spotting solution and/or the presence of waterin a overhydrated substrate results in the polymerization of silyloligonucleotides and thus interfered with the modification of thesurface with the desired molecule. Thus, dried polar aprotic solventssuch as DMF, DMSO and dried polar solvents like ethanol, isopropanol andmixture of solvents like DMF/Pyridine were found to be suitable solventsfor arraying the silyl modified oligonucleotides. The presence of water(>1%) in the spotting solution or over hydration of slidesresults inspot branching after arraying. Spot branching is undesirable because itmay lead to false positive results in binding studies.

(b) Method No. 2

As shown in FIG. 5, a method is shown for attaching multiple 5′ or 3′amino DNA molecules to a glass surface. To 1 equivalent of silyl aminein dry acetonitrile, 1.2 equivalents of tetraisocyante is added dropwiseand the reaction mixture is stirred at room temperature for 10 minutesto form compound 3. 5′ or 3′-amine linked oligonucleotide is synthesizedand deprotected using aqueous ammonia conditions by conventionalprocedures. After HPLC purification, 5′ or 3′-amine free oligonucleotideis treated with compound 3 in a 1:10 DMSO/ethanol (v/v) mixture. After10 minutes, the modified oligonucleotides are evaporated under vacuumand spotted on unmodified glass surface in DMSO or DMF media.

Example 2 Detection of Factor V Target Sequence Using a DNA Array Chip

This Example illustrates that DNA plates prepared as described inExample 1 are useful for sandwich hybridization assays for detection ofnucleic acid targets.

(a) Gold Colloid Preparation:

Gold colloids (13 nm diameter) were prepared by reduction of HAuCl₄ withcitrate as described in Frens, Nature Phys. Sci., 241, 20 (1973) andGrabar, Anal. Chem., 67, 735 (1995). Briefly, all glassware was cleanedin aqua regia (3 parts HCl, 1 part HNO₃), rinsed with Nanopure H₂O, thenoven dried prior to use. HAuCl₄ and sodium citrate were purchased fromAldrich Chemical Company. Aqueous HAuCl₄ (1 mM, 500 mL) was brought toreflux while stirring. Then, 38.8 mM sodium citrate (50 mL) was addedquickly. The solution color changed from pale yellow to burgundy, andrefluxing was continued for 15 min. After cooling to room temperature,the red solution was filtered through a Micron Separations Inc. 1 micronfilter. Au colloids were characterized by UV-Vis spectroscopy using aHewlett Packard 8452A diode array spectrophotometer and by TransmissionElectron Microscopy (TEM) using a Hitachi 8100 transmission electronmicroscope. Gold particles with diameters of 13 nm will produce avisible color change when aggregated with target and probeoligonucleotide sequences in the 10-35 nucleotide range.

(b) Synthesis of Oligonucleotides:

Oligonucleotides were synthesized on a 1 micromole scale using aMilligene Expedite DNA synthesizer in single column mode usingphosphoramidite chemistry. Eckstein, F. (ed:) Oligonucleotides andAnalogues: A Practical Approach (IRL Press, Oxford, 1991). All solutionswere purchased from Milligene (DNA synthesis grade). Average couplingefficiency varied from 98 to 99.8%, and the final dimethoxytrityl (DMT)protecting group was cleaved from the oligonucleotides to do finalepiendrosterone coupling on the synthesizer itself. Capture strands weresynthesized with DMT on procedure and purified on HPLC system.

(c) Purification of Oligonucleotides

Reverse phase HPLC was performed with using Agilent 1100 series systemequipped with Tosch Biosep Amberchrom MID-G CG-300S column(10×118 mm, 35μm particle size) using 0.03 M Et₃NH⁺ OAc⁻ buffer (TEAA), pH 7, with a1%/min. gradient of 95% CH₃CN/5% TEAA. The flow rate was 1 mL/min. withUV detection at 260 nm. The final DMT attached was deprotected on HPLCcolumn itself using 1-3% trifluoro acetic acid and TEAA buffer. Aftercollection and evaporation of the buffer contained the DMT cleavedoligonucleotides, was then evaporated to near dryness. The amount ofoligonucleotide was determined by absorbance at 260 nm, and final purityassessed by reverse phase HPLC.

The same protocol was used for epiendrosterone linked-oligonucleotidesfor probe preparation and no DMT removal needed¹⁰.

(d) Attachment of Oligonucleotides to Gold Nanoparticles

Probes used in the Example: (3′-act tta aca ata g-a₂₀-Epi-5′ (SEQ IDNO:1) and 3′-t taa cac tcg c a₂₀-Epi-5′ (SEQ ID NO:2)) were attached inthe following fashion. These probes were designed for M13 targetsequence detection.

A 1 mL solution of the gold colloids (15 nM) in water was mixed withexcess (3.68:M) 5′epi-endrosterone linked-oligonucleotide (33 and 31bases in length) in water, and the mixture was allowed to stand for12-24 hours at room temperature. Then, 100 μL of a 0.1 M sodium hydrogenphosphate buffer, pH 7.0, and 100 μL of 1.0 M NaCl were premixed andadded. After 10 minutes, 10 μL of 1% aqueous NaN₃ were added, and themixture was allowed to stand for an additional 20 hours then increasedthe salt concentration to 0.3. After standing 4 h at 0.3 M NaCl againincreased to 1M NaCl and kept further 16 h. This “aging” step wasdesigned to increase the surface coverage by the epi disulfidelinked-oligonucleotides and to displace oligonucleotide bases from thegold surface. Somewhat cleaner, better defined red spots in subsequentassays were obtained if the solution was frozen in a dry-ice bath afterthe 40-hour incubation and then thawed at room temperature. Either way,the solution was next centrifuged at 14,000 rpm in an EppendorfCentrifuge 5414 for about 15 minutes to give a very pale pinksupernatant containing most of the oligonucleotide (as indicated by theabsorbance at 260 nm) along with 7-10% of the colloidal gold (asindicated by the absorbance at 520 nm), and a compact, dark, gelatinousresidue at the bottom of the tube. The supernatant was removed, and theresidue was resuspended in about 200 μL of buffer (10 mM phosphate, 0.1M NaCl) and recentrifuged. After removal of the supernatant solution,the residue was taken up in 1.0 mL of buffer (10 mM phosphate, 0.1 MNaCl) and 10 μL of a 1% aqueous solution of NaN₃. Dissolution wasassisted by drawing the solution into, and expelling it from, a pipetteseveral times. The resulting red master solution was stable (i.e.,remained red and did not aggregate) on standing for months at roomtemperature, on spotting on silica thin-layer chromatography (TLC)plates , and on addition to 2 M NaCl, 10 mM MgCl₂, or solutionscontaining high concentrations of salmon sperm DNA. For examples 2-5 weprepared different set of Factor V probes using an aqueous solution of17nM (150 μL) Au colloids, as described above, was mixed with 3.75 μM(46 μL) 5′-epiendrosterone-a₂₀-tattcctcgcc (SEQ ID NO:3), and allowed tostand for 24 hours at room temperature in 1 ml Eppendorf capped vials. Asecond solution of colloids was reacted with 3.75 μM (46 μL)5′-epiendrosterone-a₂₀-attccttgcct-3′.(SEQ ID NO:4). Note that theseoligonucleotides are non-complementary. The residue was dissolved usingthe same procedure described above and the resulting solution was storedin a glass bottle until further use.

(e) Hybridization Conditions

Stock buffer solution: For the hybridization buffer, the following stocksolution was used: 3.0 NaCl, 0.3 M Na-Citrate, 10 mM MgCl₂, 4.0 mMNaH₂PO₄ and 0.005% SDS.

Hybridization assay was performed using diluted buffer (0.78M NaCl, 70mM sodium citate, 2.64 mM MgCl₂, 1.1 mM sodium phosphate, 0.01%) fromthe stock buffer solution by adding 0.5% of Tween. In a typicalexperiment procedure, target and probe were mixed with the hybridizationbuffer and heated the mixture at 95° C. for 5 minutes. After cooling toroom temperature aliquots were transferred on to the glass substrate andplaced in humidity chamber for hybridization (Different assays were doneat different temperature conditions since each probe has a differentmelting temperature). After hybridization, plates were washed with twodifferent wash buffers and spin dried. Plates dried were treated withsilver amplification solutions (silverA+silverB) (silver amplificationkit available from SIGMA, St.Louis, Mo. 63178, catalog no: S 5020 and S5145) and the data was collected from the amplified plates using animaging system for data collection described in (Nanosphere, Inc.assignee) U.S. patent application Ser. No. 10/210,959 andPCT/US02/24604, both filed Aug. 2, 2002, which are incorporated byreference in their entirety.

(f) Target Sequence Used

This Factor V target sequence was used in examples 2-6 for detection.M13 probes were used in example 1 for direct probe targeting to capturestrand test the plates and no target detection was performed here. Butfrom example 2-5 Factor V target detection was done in presence ofFactor V probes and M13 probes. Here M13 probes served as controls. Inplate no: 5 different combination of assay were performed on one plateincluding Factor V wild type and mismatch detection. Each well in plateno:6 was clearly defined with target and probes used.

Factor V Wild Type Sequence:

-   5′gacatcgcctctgggctaataggactacttctaatctgtaagagcagatccctggacaggcaaggaatacaggtattttgtccttgaagtaa    cctttcag 3′ (SEQ ID NO:5)    Probe Sequence:-   Probe FV (13D): 5′-Epi-a₂₀-tattcctcgcc 3′ (SEQ ID NO: 3)-   Probe FV (26D): 5′-Epi-a₂₀-attccttgcct3′ (SEQ ID NO: 4)    Capture Strand Sequence for Factor V Target Detection:-   5′-tcc tga tga aga tta gac att ctc gtc-NH—CO—NH—Si—(OEt)₃-3′ (SEQ ID    NO:6)    Stock buffer solution: For the hybridization buffer, the following    stock solution was used: 3.0 NaCl, 0.3 M Na-Citrate, 10 mM MgCl₂,    4.0 mM NaH₂PO₄ and 0.005% SDS.

Example 3 Detection of M13 Target Sequence Using DNA Array Chip

In this Example, probe was targeted directly to the capture strand and adetection assay was performed. Plates Nos. 1-3 were prepared asdescribed in Example 1 (method no. 1). In Plates 2 & 3, probes (FIG. 6)were clearly hybridized to the capture strand within 45 minutes. Thegold colloid nanoparticles hybridized to the capture were clearlyvisible before silver amplification. In plate no 1 (FIG. 6), a differentprobe was used and the assay was developed to show the specificity.After silver stain development, signals were not shown on the glasssurface even after silver amplification. This experiment established thespecificity of the DNA chip prepared in accordance with the invention.

M13 Capture sequence:

-   5′-tga aat tgt tat c-NH—CO—NH—Si—(OEt)₃-3′ (SEQ ID NO: 7)    Probe used on plates Nos. 2-3 plates:-   3′-act tta aca ata g-a₂₀-Epi-5′ (SEQ ID NO: 1)

On plate no.1, a detection probe 3′-t taa cac tcg c-a₂₀-Epi-5′ (SEQ IDNO:2) was used which was non-complementary to the capture strand forsequence specificity testing (no signals). This clearly showed thespecificity of the both capture strand sequence and the probe. In bothcases, 6 nM probe was used in diluted buffer conditions. In a typicalexperimental procedure, 30□l of the diluted buffer (1.3M NaCl, 130 mMsodium citrate, 4.38 mM MgCl₂, 1.82 mM sodium phosphate, 0.003% SDS) and20μl of probe (10 nM) was flooded on the arrayed glass chip and allowedto hybridize for 1 .5 h at room temperature. The final concentration ofprobe was 4nM and buffer concentration was 0.78M NaCl, 70 mM sodiumcitrate, 2.64 mM MgCl₂, 1.1 mM sodium phosphate, 0.002% SDS. Thereafter,the chip was washed with 0.75 M sodium chloride, 75 mM citrate and 0.05%Tween buffer and then washed again with 0.5M sodium nitrate buffer. Thenplates were treated with silver amplification solutions silver A+SilverB(1 mL+1 mL=total 2 mL) for 4 minutes and washed with nanopure water.Finally, the plates were exposed to the imaging system for datacollection as discussed above.

Example 4 Detection of Factor V Target Sequence Using a DNA Array Chip

In this Example, two different silanized capture strands were spotteddirectly on the plate and detected. The plate was prepared as describedin Example 1 (method no. 1). The middle row always carried the positivecontrol capture with other capture on top and bottom rows. Here, wildtype, mutant and heterozygous samples were used for the detection. Allsamples were showed signals in the proper place using the abovementioned assay conditions. See FIG. 7.

a) Positive Controls Capture Sequence:

(SEQ ID NO:7) 5′-tga aat tgt tat c-NH-CO-NH-Si-(OEt)₃-3′Probe Used was for Positive Control:

3′-act tta aca ata g-a₂₀-Epi-5′ (SEQ ID NO:1)b) Probes Used for Target Detection are:

Probe FV 13D (probe for wild type target): 5′-Epi-a₂₀-tattcctcgcc 3′(SEQ ID NO:3)

Probe FV 26D (probe for mutant target): 5′-Epi-a₂₀-attccttgcct3′ (SEQ IDNO:4)

Capture Strand Sequence for Factor V Target Detection:

-   5′-tcc tga tga aga tta gac att ctc gtc-NH—CO—NH—Si—(OEt)₃-3′ (SEQ ID    NO:6)    Factor V Wild Type Target Sequence:-   5′    gacatcgcctctgggctaataggactacttctaatctgtaagagcagatccctggacaggcaaggaatacaggtattttgtccttgaagtaa    cctttcag 3′ (SEQ ID NO: 5)    Mutant Factor V Target Sequence:-   gtaggactacttctaatctgtaagagcagatccctggacaggtaaggaatacaggtattttgtccttgaagtaacctttcag-3′    (SEQ ID NO:8)-   Heterozygous: 50% of wild type and 50% of mutant target.-   Well 1: Heterozygous—Probe 26D was used-   Well 2: Heterozygous—Probe 13D was used-   Well 3: Control—with probe 26D, only positive control should show up-   Well 4: Control—with probe 13D, only positive control should show up-   Well 5: Mutant—target with mutant probe 26D + positive control probe-   Well 6: Mutant target—with wild type probe 13D + positive control-   Well 7: Heterozygous—with probe 26D-   Well 8: Heterozygous—with probe 13D-   Well 9: Wild type target—with mutant probe 26D-   Well 10: Wild type target—with wild type probe 13D

Example 5 Detection of MTHFR Target Sequence on a DNA Array Plate

In this Example, an MTHFR 100 mer synthetic target and 208 base pair PCRproduct (10 nM˜50 nM) was used in the detection assay. The plates wereprepared as described in Example 1 (method no. 1). Alternative wellswere used as controls using M13 target and MTHFR 18 mer probe and didnot show even traces of silver, following silver signal amplification.As shown in plate no. 1 (FIG. 8), an experiment was performed at 70° C.to show that probe does not hybridize above melting temperature (MTHFRtarget and 18 mer probe). The results show probe specificity and that athigh temperature, the probes are not binding nonspecifically to thesilyl oligo-attached substrate.

100 mer Synthetic Target:

5′-aag cac ttg aag gag aag gtg tct gcg gga gcc gat ttc atc atc acg cag(SEQ ID NO:9) ctt ttc ttt gag gct gac aca ttc ttc cgc ttt gtg aag gcatgc acc ga-3′18 mer Probe Sequence Used on all Three Plates:

(SEQ ID NO:10) 3′-ctg tgt aag aag gcg ttt-A₂₀-Epi-5′

-   PCR product: 208 base pair-   5′ccttgaacaggtggaggccagcctctcctgactgtcatccctattggcaggttaccccaaaggccaccccgaagcagggagctttgagg    ctgacctgaagcacttgaaggagaaggtgtctgcgggagccgatttcatcatcacgcagcttttctttgaggctgacacattcttccgctttgtg    aaggcatgcaccgacatgggcatcacttgccccatcgtccccgggatctttcccatccaggtgaggggcccaggagagcccataagctcc    ctccaccccactctcaccgc (SEQ ID NO: 11)    Experimental Conditions:

In a typical experimental procedure (on plate no:2), to 30 μl of thediluted buffer (1.3M NaCl, 130 mM sodium citrate, 4.38 mM MgCl₂, 1.82 mMsodium phosphate, 0.003% SDS), 10 μl of 18 mer probe (10 nM) and 2 μl of100 mer synthetic target (10 μM) 8 μl of water were mixed and flooded onthe arrayed glass chip and allowed to hybridize for 1.5 h at roomtemperature. The final concentration of probe was 2 nM and targetconcentration was 400 pM and buffer concentration was 0.78M NaCl, 70 mMsodium citate, 2.64 mM MgCl₂, 1.1 mM sodium phosphate, 0.01%). Afterthat washed with 0.75 M sodium chloride, 75 mM citrate and 0.05% Tweenbuffer and then washed again with 0.5M sodium nitrate buffer. After thatplates were treated with silver A+SilverB (1 mL+1 mL=total 2 mL) (silveramplification kit available from SIGMA, St. Louis, Mo. 63178, catalogno: S 5020 and S 5145) for 4 minutes and washed with nanopure water.Finally plates were exposed to imaging system for data collection asdiscussed above. In Example 3 on plate no:2, wells no: 2 1,4, 5, 8 arecontrols and controls made up with M13 synthetic target and MTHFR 18 merprobe (5′-tat gct tcc ggc tcg tat gtt gtg tgg aat tgt gag cgg ata acaatt tca-3′). (SEQ ID NO: 12)

As mentioned earlier, the experiment on plate no.1 (FIG. 8) wasperformed at 70° C. to show that above melting temperature probe 18 merprobe did not bind to the capture probe.

Plate no.3 (FIG. 8) was generated following the same experimentalprocedure and using the same probes. 10 μl (2 nm˜10 nM) of MTHFR PCRproduct was used as target. Plate no.3 wells 2, 3, 6 and 7 are thecontrols with Factor V 99 mer mutant target and MTHFR 18 mer probe.

Factor V 99 mer Mutant Factor V Target had the Following Sequence:

-   5′gtaggactacttctaatctgtaagagcagatccctggacaggtaaggaatacaggtattttgtccttgaagtaacctttcag-3′)    (SEQ ID NO: 8)

EXAMPLE 6 Detection of Factor V Target Sequence on DNA Array Plate

In this Example and in the following Example 7, the same capture strandswere arrayed on the plate. The purpose of this experiment was to findout the difference in intensity of the spots after silver developmentwhen same oligomer was spotted on the slide at different places.Positive control was spotted in the middle of two Factor V 4G oligomercaptures on the slide. The results are shown in FIG. 9.

Capture Strand Sequence for Factor V Target Detection was:

5′ tcc tga tga aga tta gac att ctc gtc-NH-CO-NH-Si-(OEt)₃-3′ (SEQ IDNO:6)Positive Capture Control Capture Spotted was (M13):

(SEQ ID NO:7) 5′ tga aat tgt tat c-NH-CO-NH-Si-(OEt)₃-3′The Target Sequence Used was Wild Type Factor V 99base Pair SingleStrand DNA having the Following Sequence:

-   gtaggactacttctaatctgtaagagcagatccctggacaggcaaggaatacaggtattttgtccttgaagtaacctttcag-3′)    (SEQ ID NO: 13)    Mutant Factor V Target had the Following Sequence:-   gtaggactacttctaatctgtaagagcagatccctggacaggtaaggaatacaggtattttgtccttgaagtaacctttcag-3′)    (SEQ ID NO:8)    and Probes Used had the Following Sequence:-   probe FV 13D: 5′-Epi-a₂₀-tattcctcgcc 3′ (SEQ ID NO:3),-   probe FV 26D: 5′-Epi-a₂₀-attccttgcct3′ (SEQ ID NO:4).    Capture Strand Sequence for Factor V Target Detection:

5′-tcc tga tga aga tta gac att ctc gtc-NH-CO-NH-Si-(OEt)₃-3′ (SEQ IDNO:6)Positive control sequence: 5′-tga aat tgt tat c-NH₂-3′ (SEQ ID NO: 14)and probe used for positive control was: 3′-act tta aca ata g-a₂₀-Epi-5′(SEQ ID NO: 1)

In a typical experimental procedure, to 25 μl of the diluted buffer(1.3M NaCl, 130 mM sodium citrate, 4.38 mM MgCl₂, 1.82 mM sodiumphosphate, 0.003% SDS), 10 μl of probe (10 nM) and 10 μl of PCR target(15-50 nM) and 5 μl of positive control probe (10 nM) were mixed andflooded on the arrayed glass chip and allowed to hybridize for 1.5 h atroom temperature. The final concentration of probe was 2 nM, and bufferconcentration was 0.78M NaCl, 70 mM sodium citate, 2.64 mM MgCl₂, 1.1 mMsodium phosphate, 0.01%). that the plates was then washed with 0.75Sodium chloride, 75 mM citrate and 0.05% tween buffer and then washedagain with 0.5M Sodium Nitrate buffer. The plates were treated withsilver A+SilverB (1 mL+1 mL=total 2 mL) for 4 minutes and washed withnanopure water. Finally, the plates were exposed to the imaging systemdescribed above for data collection. Both positive control probe andtarget reacted probe were mixed and the assay was run to show theselectivity of the probe. The wells were identified as follows:

-   Wells 1, 6, 8 and 9 have only positive control probe with target and    buffer.-   Wells 2, 5 had both positive control probe and target probe with    targets and buffer.-   Wells 4, 7 and 10 have only target probe with target and buffer and    here positive control probe and target were absent.-   Well 3 did not have any target and positive control probe but it had    target probe and buffer.    These results (FIG. 9) show that probes were specific to target    detection and no non-specific background noise was observed when    target was absent.

EXAMPLE 7 Detection of Factor V Target Sequence

In this Example, all capture strands pattern is the same as described inExample no.6. Moreover, the same experimental conditions andconcentrations described in Example 6 were used to perform the assay at52° C. Wild type and mutant targets were given in the example 6. Theresults are shown in FIG. 10. The wells are identified as follows:

-   Well 1: Positive control probe directly probing to the capture    strand in the same buffer conditions mentioned in example 4.-   Well 2: Factor V Probe 5′-Epi-a₂₀-attccttgcct-3′ (26D) (SEQ ID    NO: 4) and Factor V 99base pair mutant target, positive control    probe and buffer.-   Well 3: Factor V Probe 5′-Epi-a₂₀-attccttgcct-3′ (26D) (SEQ ID    NO: 4) and Factor V 99base pair mutant target and hybridization    buffer.-   Well 4: Probe 13D and Factor V mutant PCR target, positive control    and hybridization buffer.-   Well 5: Probe 13D and Factor V mutant PCR target, and hybridization    buffer.-   Well 6: Control (MTHFR target and Probe 13D and hybridization    buffer).-   Well 7: Wild type Factor V target, probe (26D), positive control    probe and hybridization buffer,-   Well 8: Wild type Factor V target and probe (26D), and hybridization    buffer.-   Well 9: Wild type Factor V target, probe 13(D), positive control    probe and hybridization buffer.-   Well 10: Wild type Factor V target, probe 13(D), and hybridization    buffer.-   Probe FV 13D: 5′-Epi-a₂₀-tattcctcgcc-3′ (SEQ ID NO: 3)-   Probe FV 26D: 5′-Epi-a₂₀-attccttgcct-3′ (SEQ ID NO: 4)    These results (FIG. 10) show that probes were reacted specifically    to the target and there is no cross hybridization between probes and    targets were observed when probes were mixed with different targets.

REFERENCES

-   1. Nucleic Acids research, vol 22, 5456-5465 (1994).-   2. Nucleic Acids research, vol 24, 3040-3047 (1996).-   3. Nucleic Acids research, vol 24, 3031-3039 (1996).-   4. Nucleic Acids research, vol 27, 1970-1977 (1999).-   5. Angew. Chem. Int. Ed, 38, No.9, 1297(1999)-   6. Analytical biochemistry 280, 143-150 (2000).-   7. (a) Nucleic Acids research, vol. 28, No.13 E71 (2000);    -   (b) Huber et al. WO 01/46214, published Jun. 28, 2001    -   (c) Huber et al. WO 01/46213, published Jun. 28, 2001    -   (d) Huber et al. WO 01/46464, published Jun. 28, 2001-   8. Nucleic Acids research, vol 29, 955-959 (2001).-   9. Nucleic Acids research, vol 29, No.13 e69 (2001).-   10. Bioconjugate Chemistry, 2000, 11, 289-291

1. A method for immobilizing a molecule onto a surface, said methodcomprising the steps of: (a) contacting Si(NCY)₄ with an agent having aformula ii:(R₁)(R₂)(R₃)Si—X-Z  ii so as to form a first reactive intermediate,wherein Y represents oxygen or sulphur, R₁, R₂ and R₃ independentlyrepresents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted withone or more groups selected from the group consisting of C₁-C₆ alkyl andC₁-C₆ alkoxy; X represents linear or branched C₁-C₂₀ alkyl or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or moreheteroatoms comprising oxygen, nitrogen, or sulfur; and Z represents ahydroxy or amino group, with the proviso that at least one of R₁, R₂ orR₃ represents C₁-C₆ alkoxy; (b) contacting the first reactiveintermediate with a molecule so as to form a second reactiveintermediate; (c) contacting the second reactive Intermediate with saidsurface so as to immobilized the molecule onto said surface.
 2. Themethod of claim 1 wherein the surface is a glass surface.
 3. The methodof claim 1 wherein the surface has at least one group that reacts withthe reactive intermediate.
 4. The method of claim 3 wherein the groupcomprises a hydroxyl or amino group.
 5. The method of claim 1 whereinthe agent is 4-aminophenyl trimethoxysilane, 4-aminophenyltilethoxysilane, 3-aminophenyltrimethoxysilane,3-amenophenyltriethoxysilane, or mixtures thereof.
 6. The method ofclaim 5 wherein the agent and tetraisocyanatosilane is present at a 4:1molar ratio.
 7. The method of claim 1 wherein the molecule and the firstreactive intermediate is present in a 3:1 molar ratio.
 8. The method ofclaim 1 wherein Si(NCY)₄ and the agent are present at a 1:1 molar ratioand the first reactive Intermediate has a formula iv:(R₁)(R₂)(R₃)Si—X-Z-CYNH—Si(NCY)₃  iv wherein R₁, R₂ and R₃ independentlyrepresents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted withone or more groups selected from the group consisting of C₁-C₆ alkyl andC₁-C₆ alkoxy; X represents linear or branched C₁-C₂₀ alkyl or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or moreheteroatoms comprising oxygen, nitrogen, or sulfur; Y represents oxygenor sulfur; and Z represents oxygen or NH, with the proviso that at leastone of R₁, R₂, or R₃ represents C₁-C₆ alkoxy.
 9. The method of claim 8wherein R₁, R₂ and R₃ represents methoxy, X represents phenyl, Yrepresents oxygen, and Z represents NH.
 10. The method of claim 8wherein the second reactive intermediate has a formula v:(R₁)(R₂)(R₃)Si—X-Z-CYNH—Si(NHCYL-M)₃  v wherein R₁, R₂ and R₃independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy; L represents a linking group; Xrepresents linear or branched C₁-C₂₀ alkyl or aryl substituted with oneor more groups selected from the group consisting of C₁-C₆ alkyl andC₁-C₆ alkoxy, optionally substituted with one or more heteroatomscomprising oxygen, nitrogen, or sulfur; Y represents oxygen or sulfur;and Z represents oxygen or NH; and M represents a molecule, with theproviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy. 11.The method of claim 10 wherein L represents a nucleophilic group fromthe molecule.
 12. The method of claim 11 wherein the nucleophilic groupcomprises —NH, —S—, —O—, or —OOC—.
 13. The method of claim 11 whereinSi(NCY)₄ and the agent are present at a 1:2 molar ratio and the firstreactive intermediate has a formula vi:[(R₁)(R₂)(R₃)Si—X-Z-CYNH]₂—Si(NCY)₂  vi wherein R₁, R₂ and R₃independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy; X represents linear or branched C₁-C₂₀alkyl or aryl substituted with one or more groups selected from thegroup consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substitutedwith one or more heteroatoms comprising oxygen, nitrogen, or sulfur, Yrepresents oxygen or sulfur; and Z represents oxygen or NH, with theproviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy. 14.The method of claim 13 wherein R₁, R₂ and R₃ represents methoxy, Xrepresents phenyl, Y represents oxygen, and Z represents NH.
 15. Themethod of claim 13 wherein the second reactive intermediate has aformula vii:[(R₁)(R₂)(R₃)Si—X-Z-CYNH]₂Si[NHCYL-M]₂  vii wherein R₁, R₂ and R₃independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy; L represents a linking group; Xrepresents linear or branched C₁-C₂₀ alkyl or aryl substituted with oneor more groups selected from the group consisting of C₁-C₆ alkyl andC₁-C₆ alkoxy, optionally substituted with one or more heteroatomscomprising oxygen, nitrogen, or sulfur; Y represents oxygen or sulfur:and Z represents oxygen or NH; and M represents a molecule, with theproviso that at least one of R₁, R₂, or R₃ represent C₁-C₆ alkoxy. 16.The method of claim 15 wherein L represents a nucleophilic group fromthe molecule.
 17. The method of claim 16 wherein the nucleophilic groupcomprises —NH, —S—, —O—, or —OOC—.
 18. The method of claim 1 wherein themolecule comprises a probe.
 19. The method of claim 18, wherein theprobe comprises a protein, a peptide, a nucleic acid, a peptide nucleicacid, an amino acid, a linked nucleic acid, a nucleoside triphosphate, acarbohydrate, a lipid, a lipid bound protein, an aptamer, a virus, acell fragment, or a whole cell.
 20. The method of claim 19, wherein thelipid bound protein comprises a G-protein coupled receptor.
 21. Themethod of claim 18, wherein the probe comprises an antibody, an antigen,a receptor, or a ligand.
 22. A method for immobilizing a molecule onto aglass surface, said method comprising the steps of: (a) contactingSi(NCY)₄ with an agent having a formula ii:(R₁)(R₂)(R₃)Si—X-Z  ii so as to form a first reactive intermediate,wherein Y represents oxygen or sulfur; R₁, R₂ and R₃ independentlyrepresents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted withone or more groups selected from the group consisting of C₁-C₆ alkyl andC₁-C₆ alkoxy; X represents linear or branched C₁-C₂₀ alkyl or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or moreheteroatoms comprising oxygen, nitrogen, and sulfur; and Z represents ahydroxy or amino group, with the provision that at least of one R₁, R₂,or R₃ represents C₁-C₆ alkoxy; (b) contacting the first reactiveintermediate with a molecule so as to form a second reactiveintermediate; (c) contacting the second reactive intermediate with saidsurface so as to immobilized the molecule onto said surface.
 23. Themethod of claim 22 wherein the surface has at least one group thatreacts with the reactive intermediate.
 24. The method of claim 23wherein the group comprises a hydroxyl or amino group.
 25. The method ofclaim 22 wherein the agent is 4-aminophenyl trimethoxysilane,4-aminophenyl trimethoxysilane, 3-aminophenyltriethoxysilane,3-aminophenyltriethoxysilane, or mixture thereof.
 26. The method ofclaim 22 wherein the agent and tetraisocyanatosilane is present at a 1:1molar ratio.
 27. The method of claim 22 wherein the molecule and thefirst reactive intermediate is present in a 3:1 molar ratio.
 28. Themethod of claim 22 wherein Si(NCY)₄ and the agent are present at a 1:1molar ratio and the first reactive intermediate has a formula iv:(R₁)(R₂)(R₃)Si—X-Z-CYNH—Si(NCY)₃  iv wherein R₁, R₂ and R₃ independentlyrepresents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted withone or more groups selected from the group consisting of C₁-C₆ alkyl andC₁-C₆ alkoxy; X represents linear or branched C₁-C₂₀ alkyl or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or moreheteroatoms comprising oxygen, nitrogen, or sulfur; Y represents oxygenor sulfur: and Z represents oxygen or NH, with the proviso that at leastone of R₁, R₂, or R₃ represents C₁-C₆ alkoxy.
 29. The method of claim 28wherein R₁, R₂ and R₃ represents methoxy, X represents phenyl, Yrepresents oxygen, and Z represents NH.
 30. The method of claim 28wherein the second reactive intermediate has a formula v:(R₁)(R₂)(R₃)Si—X-Z-CYNH—Si(NHCYL-M)₃  v wherein R₁, R₂ and R₃independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy; L represents a linking group; Xrepresents linear or branched C₁-C₂₀ alkyl or aryl substituted with oneor more groups selected from the group consisting of C₁-C₆ alkyl andC₁-C₆ alkoxy, optionally substituted with one or more heteroatomscomprising oxygen, nitrogen, or sulfur; Y represents oxygen or sulphur,and Z represents oxygen or NH; and M represents a molecule, with theproviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy. 31.The method of claim 30 wherein L represents a nucleophilic group fromthe molecule.
 32. The method of claim 31 wherein the nucleophilic groupcomprises —NH, —S—, —O—, or —OCC—.
 33. The method of claim 22 whereinSi(NCY)₄ and the agent are present at a 1:2 molar ratio and the firstreactive intermediate has a formula vi:[(R₁)(R₂)(R₃)Si—X-Z-CYNH]₂—SI(NCY)₂  vi wherein R₁, R₂ and R₃Independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy; X represents linear or branched C₁-C₂₀alkyl or aryl substituted with one or more groups selected from thegroup consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substitutedwith one or more heteroatoms comprising oxygen, nitrogen, or sulfur; Yrepresents oxygen or sulfur; and Z represents oxygen or NH, with theproviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy. 34.The method of claim 33 wherein R₁, R₂ and R₃ represents methoxy, Xrepresents phenyl, Y represents oxygen, and Z represents NH.
 35. Themethod of claim 33 wherein the second reactive intermediate has aformula vii:[(R₁)(R₂)(R₃)Si—X-Z-CYNH]₂Si(NHCYL-M)₂  vii wherein R₁, R₂ and R₃independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or arylsubstituted with one or more groups selected from the group consistingof C₁-C₆ alkyl and C₁-C₆ alkoxy; L represents a linking group; Xrepresents linear or branched C₁-C₂₀ alkyl or aryl substituted with oneor more groups selected from the group consisting of C₁-C₆ alkyl andC₁-C₆ alkoxy, optionally substituted with one or more heteroatomscomprising oxygen, nitrogen, or sulfur; Y represents oxygen or sulfurand Z represents oxygen or NH; and M represents a molecule, with theproviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy. 36.The method of claim 35 wherein L represents a nucleophilic group fromthe molecule.
 37. The method of claim 36 wherein the nucleophilic groupcomprises —NH, —S—, —O—, or —OOC—.
 38. The method of claim 22 whereinthe molecule comprises a probe.
 39. The method of claim 38, wherein theprobe comprises a protein, a peptide, a nucleic acid, a peptide nucleicacid, an amino acid, a linked nucleic acid, a nucleoside triphosphate, acarbohydrate, a lipid, a lipid bound protein, an aptamer, a virus, acell fragment, or a whole cell.
 40. The method of claim 39, wherein thelipid bound protein comprises a G-protein coupled receptor.
 41. Themethod of claim 22, wherein the probe comprises an antibody, an antigen,a receptor, or a ligand.